Instrument: Illumina HiSeq 2500
Strategy: OTHER
Source: GENOMIC
Selection: other
Layout: PAIRED
Construction protocol: To profile for open chromatin, we used the Assay for Transposase Accessible Chromatin (ATACseq) protocol, performed according to Buenrostro et al., 2015 with minor modifications. The dissected samples were incubated in trypsin 0.5%/EDTA 1X at 37 °C for 10 minutes and rinsed in DMEM 1X/ FBS 10%. Cranial neural crest cells (CNCCs) were then dissociated with a pipette, filtered and collected by FACS. 50.000 CNCCs were used per transposition reaction. After sorting, CNCCs were pelleted by centrifugation (500 g for 5 minutes at 4°C), washed with cold 1X PBS buffer and pelleted again by centrifugation (500 g for 5 minutes at 4°C). They were then re-suspended in cold lysis buffer (10mM Tris-HCl pH 8.0; 10mM NaCl; 3mM MgCl2; 0.5% NP40) and pelleted by centrifugation (5 minutes, 500 g at 4°C). The supernatant was discarded. The transposition reaction was performed as reported in Buenrostro et al., 2015, steps 6 – 12, using the Nextera Sample preparation kit from Illumina. PCR Amplification was done according to Buenrostro et al., 2015, steps 13 – 18. The indexing PCR primers mentioned in the supplemental material from Buenrostro et al., 2013 were used. Amplified libraries were cleaned up using AMPure XP beads in a DNA / beads ratio of 1 / 1.8. The Quality of the libraries and size distribution was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies). Sequencing was performed on an Illumina HiSeq 2500 machine (50 bp read length, paired-end) according to Illumina standards.